Gene transfer in intact mammals

ABSTRACT

Methods and compositions are provided for gene transfer to intact mammals with expression of the exogenous genetic material in the host. Mammalian host cells which are regenerative, normally highly proliferative or subject to induced proliferation, are transformed or modified in vitro with DNA capable of replication and expression in the host cell, wherein the DNA becomes incorporated into the cell. The modified cells are found to regenerate in the host with expression of the introduced DNA. Particularly, mammalian cells were modified with genes providing for overproduction of a particular enzyme. The modified cells were reintroduced in the host under conditions providing for selective advantage of the modified cells.

The invention described herein was made in the course of, or under, agrant from the United States Public Health Service.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation application of pending patent application Ser.No. 134,234, filed Mar. 26, 1980, now U.S. Pat. No. 4,396,601.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The discovery that one could introduce exogenous genes into a bacterialhost in vitro and observe expression of the exogenous genes in thebacterial host opened up vistas of new capabilities for the productionof a wide range of compounds, particularly proteins, improved methods oftreating waste, novel types of fertilizers, and new vaccines. Whiletransformation of prokaryotes offer many new and yet envisagedopportunities, there is also great interest in being able to modifyeukaryotes and particularly mammalian cells.

Many diseases are genetically related involving genetic deficiencies,which are usually either failure to produce a gene product or productionof an abnormal product. Other situations involve treatment of a hostwith drugs which may have substantial toxicity to host cells. In theseinstances, it would be desirable to provide the host with the missingcapability, the normal capability or a defense mechanism against thedetrimental effects of the drug. The capability to modify a host'sgenetic structure to provide for either additional genetic capabilitiesor reparation of a defective capability on a temporary or permanentbasis opens up wide avenues in the treatment of genetic deficiencies anddisease.

2. Description of the Prior Art

Methods of introducing genetic material into a host cell include viralvectors Munyon et al. J. Virol. 7:813-820, 1971; cell-cell fusion, thefusion to cells of a limited number of chromosomes enveloped in nuclearmembranes, Fournier et al. Proc. Natl. Acad. Sci. 74:319-323, 1977; andcellular endocytosis of microprecipitates of calcium-DNA complex,Bachetti and Graham, ibid. 74:1590-1594, 1977; Maitland and McDougall,Cell 11:233-241, 1977; Pellicer et al. ibid. 14:133-141, 1978 and Wigleret al. ibid. 14:725-731, 1978. Cell lines lacking thymidine kinase arereadily transformed by appropriate DNA to a tk⁺ status when grown in thepresence of a folic acid inhibitor and thymidine. Pellicer, supra andWigler, supra.

SUMMARY OF THE INVENTION

Methods and compositions are provided for providing mammalian hosts withadditional genetic capability, either a novel capability or enhancementof an existing one. Host cells capable of regeneration are removed andtreated with genetic material under conditions whereby the geneticmaterial is introduced into the host cells and becomes capable ofreplication and expression. The introduced genetic material includes atleast one marker which allows for selective advantage for the host cellsin which the introduced genetic material is capable of expression. Thehost cells are returned to the host under regenerative conditions,preferably of rapid proliferation of the cells, optionally withstressing of the host to provide a selective advantage for thegenetically modified cells. It is found under these conditions, that themodified cells proliferate and express the genetic material which wasintroduced. Particularly, genetic material was employed which providedfor expression of an enzyme. Either under the normal conditions of thehost or subjecting the host to an enzyme antagonist, a selectiveproliferative advantage for the modified cells having overproduction ofthe enzyme resulted, in contrast to the normal cells incapable of suchoverproduction. By use of this approach, animals were obtained in whichthe majority of the type of cells involved contained the added geneticmaterial in a functionally active state.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

In accordance with the subject invention, a host is genetically modifiedby removing from the host or syngeneic source cells capable ofregeneration when present in the host. The cells are then combined withDNA having genes capable of expression to provide a selective advantagefor cells, under conditions where cells incorporate the DNA. The cells,which will include cells having the additional DNA, are then returned tothe host. The genes providing the selective advantage can be combinedwith other genetic material which will be incorporated in conjunctionwith the gene supplying the selective advantage. The gene providing theselective advantage will be referred to as the selective marker.

Various methods may be employed for introduction of the geneticmaterial, each of the methods having advantages and disadvantages. Afterintroduction of the treated cells into the host, conditions aremaintained in the host naturally, by administration of a physiologicallyactive compound, or by dietary exclusion, to provide a selectiveadvantage for the cells which have been genetically modified. In thisway, genetic functions can be provided for a variety of purposesincluding treatment of genetic deficiencies, which includes providing agenetic capability which the host lacks or production of a normalproduct where the host produces an abnormal one; production of enzymeswhich can protect the host from cytotoxic agents; or for production of awide variety of proteins e.g. hormones, globulins or the like.

In describing the invention, the host and host cells will be consideredfirst, followed by the genetic material which may be employed formodifying the host cells and the manner in which the host cells aremodified, and concluding with the regeneration of the modified cells andthe purposes and effect of expression of the genetic material introducedinto the modified cells.

HOST AND HOST CELLS

Various mammalian hosts may be treated in accordance with the subjectinvention, such as homo sapiens and domestic animals, particularlybovine, equine, ovine and procine. The type of host cell which will beemployed is one which is capable of regeneration, preferably rapidproliferation, either naturally or induced; can be isolated from thehost or syngeneic source; can be modified by introduction of geneticmaterial, which genetic material will then be capable of expression andreplication; can be maintained in vitro, so as to be returned to thehost in a viable state; are capable of being returned to the source inthe host; and can provide the added genetic function in a form which isuseful to the host.

Among potential cells which may be employed are bone marrow cells,particularly stem cells which provide hematopoietic functions. Otherexamples of tissues which have persistent stem cells include theintestinal mucosa and the germ line tissues. Use of these techniques tointroduce genes into germ line cells may be of especial interest inbreeding improved strains of domestic animals. Other cells which can beemployed include cells of regenerative organs e.g. liver. Any bodymember which is regenerative or can be induced to regenerate can be asource of cells.

Bone marrow cells chosen for modification should optimally bepopulations rich in stem cells. Furthermore, the cells chosen arepreferably dividing, rather than stationary cells. To increase thefraction of these types of cells, the host may be treated by varioustechniques to increase the level of proliferating cells. For example,vinca alkaloids may be employed which inhibit mitosis, followed by radidproliferation of the cells.

A wide variety of genetic material (DNA) may be employed to provide forthe selective marker. The selective marker will allow for rapidproliferation of the modified cells in the host under normal conditionsof the host or where rapid proliferation is subject to inhibition. Theinhibition can be as a result of introduction of a drug which inhibits(a) proliferation because of interference with transcription of DNA ortranslation of RNA, that is, expression of one or more genes; (b) cellmembrane formation; (c) cell wall formation; (d) enzyme activity; or (e)combinations thereof.

A wide variety of drugs are known which are employed for the treatmentof disease which inhibit cell replication, so as to favor the hostagainst a parasitic invader such as bacteria, protozoa, or even aneoplastic variant of the host cell. The effectiveness of the drug maybe inhibited in a cell by introducing into the cell genes which expressan enzyme which reacts with the drug to deactivate it, genes whichoverproduce an enzyme involved in the metabolic pathway which the druginhibits, so as to provide a selective advantage for the cells havinghigher concentrations of the enzyme(s), or genes which would provide fora metabolic pathway less affected by the drug, than the endogenousmetabolic pathway.

Alternatively, the enzyme can provide for increased production of ametabolite essential to mitosis e.g. a metabolite on the biosyntheticpathway to DNA or RNA, for example, the formation of nucleosides. Themodified cells having the selective marker which provides for enhancedenzyme production permits the modified cells to compete more effectivelyfor a limited amount of metabolite precusor against the wild type cell.

The genetic material which is employed for recombination with the hostcells may be either naturally occurring, synthetic, or combinationsthereof. Depending upon the mode employed for introduction, the size ofthe genetic material introduced will vary. Furthermore, when two or moregenes are to be introduced they may be carried on a single chain, aplurality of chains, or combinations thereof. Restrictions as to thesize of a DNA fragment will be as a result of limitations due to thetechnical aspects of the vector: if a recombinant DNA is to be used, bythe packaging requirements of a viral vector; the probability oftransfer into the recipient cells by the method employed; the manner ofpreparation and isolation of the DNA fragments; or the like.

The selective markers employed can be chosen to deactivate anantimetabolite to mammalian cells, by reacting with the antimetaboliteand modifying the antimetabolite to an ineffective product. Variousenzymes and their genes are known and have been isolated fordeactivating drugs. The most numerous examples are bacterial enzymeswhich deactivate antibiotics, such as those enzymes which conferresistance to aminoglycosides and polymyxines (streptomycin, kanamycin,neomycin, amikacin, gentamicin, tobramycin, etc.), and the like. Anotherdrug which may find use is PALA. Where the drug does not provide aselective advantage, since the host metabolic pathways are not involved,a gene providing resistance to such a drug would not be useful.Illustrative of this situation are sulfonamides, which block a bacterialpathway, but not a mammalian metabolic pathway.

Alternatively, rather than providing a gene which expresses an enzyme,one could provide a gene which is not subject to interference by thedrug. For example, one could employ DNA having a mutation at the site atwhich the drug binds or DNA which results in RNA or a protein, whichsubstantially reduces the binding of the drug to the site at which thedrug is active. Illustrative of drugs which are active by binding tospecific sites are the macrolides, e.g. erythromycin andaminoglycosides, e.g. streptomycin.

The next group of drugs are chemotherapeutic agents. Protection of thehost cells from the chemotherapeutic agents may be provided byintroducing genes which overproduce the enzyme inhibited by the drug ordeactivate the drug. Illustrative drugs include methotrexate, whichinhibits dihydrofolate reductase, purine analogs, which interfere withthe enzymes involved with inosinic acid, and pyrimidine analogs, such asfluorouracil, which inhibits thymidine monophosphate synthesis.

The selective marker may provide for enhanced production of one or moremetabolites involved in proliferation, for example, production ofnucleotides or nucleosides. An illustrative gene is the gene which codesfor thymidine kinase, which is involved in the biosynthetic pathway tothymidylic acid. This selective advantage need not be associated withantimetabolite administration to the host.

In some genetic diseases the gene which corrects the genetic defect mayitself confer a replicative advantage. For example, the insertion ofgenes for adenosine deaminase into cells of the marrow of certainpatients with combined immunodeficiency disease may confer a selectiveadvantage upon the replication of their stem cells leading to theproduction of a large population of immunocompetent cells which willameliorate the effects of the disease.

Finally, one may employ genes which provide for production of a proteinother than an enzyme, which allows for selective advantage of themodified cells. For example, this can be as a result of production ofinducer which prevents repression of translation to providesemiconstitutive or constitutive production of an enzyme. In such casesa regulator gene may confer selective advantage even when no drug isemployed.

In summation, the types of DNA which will be employed for selectivemarkers include genes which react with drugs which interfere withregeneration so as to destroy activity of the drug; genes which providesites which are not susceptible to drug action, so as to prevent thedrug's action in the particular cell; genes which are repetitive forproduction of a desired protein e.g. an enzyme, which is inhibited bythe drug; or genes which affect the regulatory function of the cell, soas to provide for overproduction of a particular enzyme by the naturalprocesses of the cell, and which increase the normal replication of thecell genes to enable the cell to better compete for limited resourceswithin the body.

If a drug is to be employed for providing the selective advantage thegene employed must be appropriately related to the drug. The particulardrugs employed must be considered as to level of toxicity and effect onthe particular tissue which is being modified. Also to be considered isthe purpose of the modification, which may limit the involved drug. Inother cases the appropriate selective marker may be related tocorrection of the genetic deficiency involved with the disease or mayalter the cells proliferation in any of various ways.

A number of ways have been developed for insertion of genetic materialsinto cells. Included among these techniques are viral vectors, Munyon etal., supra; cell-cell fusion involving the fusion to cells of a limitednumber of chromosomes enveloped in nuclear membranes, Fournier andRuddle, supra; cellular endocytosis of microprecipitates of calcium-DNAcomplex, Bachetti and Graham, supra, Maitland and McDougall, supra,Pellicer et al., supra and Wigler et al., supra: minicell fusion; fusionwith liposomes containing DNA; fusion with bacterial protoplastscontaining plasmid DNA; and fusion with erythrocyte ghosts packaged withDNA. Each of the techniques has advantages and disadvantages, such asefficiency of information insertion, selectivity as to the particularnature or information of the DNA, permissible size of the DNA fragment,and the like.

When employing the microprecipitates of calcium-DNA complex, the DNAemployed may provide for a single gene, a single set of genes, e.g. thebeta-globin gene cluster, or a plurality of unrelated genes. Aspreviously indicated, the size of the DNA fragments will vary, dependingupon the particular manner used to introduce the genetic information.The mixtures of DNA which are not covalently linked may be introduced bycongression, that is, different fragments of DNA will frequentlyconcurrently enter a suspectible cell, so that those cells which havethe selective marker are also likely to have the genetic capability ofthe additional genes.

The presence of a selective marker allows for selective pressure forpreferential regeneration of the modified cell. Thus, in situationswhere gene deficiencies exist which would not provide for selectiveadvantage of a modified cell, the selective marker affords thiscapability. With bone marrow cells, the cells could be modified byintroducing genes which would provide for the correction of geneticdeficiencies, by expression of products in which the host is deficientor provide for a wild type gene for correct expression of a protein.With bone marrow stem cells, genes could be provided with the correctsequence to correct hemoglobinopathies, such as sickle cell disease andthalassemia. Other defects could include defects in the production ofplasma coagulation factors, e.g. fibrinogen, prothrombin and the variousFactors, especially Factors VIII and IX. By introducing genes providingfor structurally normal proteins fulfilling these functions, inconjunction with the ability to provide selective pressures for themodified cells, the modified cells may be maintained in the host of ahigh level for extended periods of time.

Depending upon the nature of the cells, the cells may be introduced intothe host in various ways. With bone marrow or liver cells, the cells maybe introduced intravenously. It may be desirable to treat the host toreduce the relevant cell population so that rapid cell replication willbe favored. Various techniques can be employed to achieve this result,such as the use of mitotic inhibitors, e.g. vinca alkaloids, irradiationwith X-rays, or other technique. It is desirable that prior to theintroduction of the modified cells to the host, the host have a lowlevel of the relevant cell type so that after introduction, there may bea rapid and expanding proliferation of the modified cells.

After introduction of the modified cells into the host, the host will bestressed with relevent drug(s) if these are to be employed to provideselective pressure for the modified cells. Appropriate levels of thedrug may be maintained to insure proliferation of the desired cells.Depending upon the drug, the nature of the cells, and the concerns withrepetitive introduction of modified host cells, the drug treatment maybe of relatively short or long term duration. It is found that evenafter termination of the treatment with the drug providing the selectivepressure, the cells continue to proliferate and may be maintained at ahigh level for extended periods of time.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

The following is a flow chart of the progress of the experimentation:

                  FIG. 1                                                          ______________________________________                                        Day -3  Day 0              Day 77                                             ______________________________________                                                Isolate   Mix T6T6 and Ca                                                                            Treat with Mtx                                 Pretreat                                                                              marrow    1:1 and inject                                                                             at intervals                                   marrow →                                                                       from →                                                                           cells into →                                                                           ↓                                            donor mice:                                                                             X-rayed CBA/Ca                                                                                ↓                                                      mice            ↓                                    donors  CBA/T6T6  CBA/Ca     (1) Analyze karyotype                            with      ↑   ↑  (2) Transfer marrow                              VLB       ↑   ↑      to secondary                                           ↑   ↑      irradiated CBA/Ca                                    Transform Mock trans-    mice                                                 with      formation         ↓                                          Mtx.sup.R DNA                                                                           with "wild        ↓                                                    type" DNA         ↓                                                                   Mtx treatment                                                                    ↓                                                                      ↓                                                                   Analyze karyotypes                                                            DHFR levels                                                                   Hematologic status                           ______________________________________                                    

Transformation of Mouse Bone Marrow In Vitro

Mouse fibroblast Swiss 3T6 cells highly resistant to Mtx and containingreiterated structural genes specifying DHFR were employed (See Kellemset al. J. Biol. Chem. 254:309-318., 1979). They were maintained in4×10⁻⁴ M methotrexate (Mtx) and designated 3T6 R1. DNA was isolated from3T6 R1 and from non-resistant (wild type) mouse cell lines including 3T6(fibroblastic) and L1210 (lymphocyctic leukemia) and in laterexperiments from salmon sperm (Sigma). The relative ratio ofdihydrofolate reductase synthesis and number of gene copies in 3T6 R1and 3T6 was approximately 30 to 1. DNA coprecipitated with calciumphosphate was used to transform wild type L1210 cells to methotrexateresistance by the method of Bachetti and Graman, supra, as modified byWigler et al. supra.

Equal numbers of CBA/Ca and CBA/H-T6T6 mice were injectedintraperitoneally with 3 or 4 mg/kg of the mitotic inhibitor vinblastine3 days before marrow was removed for in vitro transformation. Mitoticinhibition by this treatment is followed by a burst of proliferation.Assays of colony-forming cells (CFU-S), when compared with total cellcounts, showed that suspensions from animals thus treated wererelatively depleted of mature cells and enriched approximately 3-fold inpluripotent spleen colony-forming cells (CFU-S). On the day oftransformation (designated day 0, FIG. 1) single cell suspensions inMcCoy's 5A medium with 15% fetal calf serum were obtained from femursand tibias of sacrificed animals.

Cells from Ca and T6T6 animals were placed in separate pools. All T6T6animals had the characteristic marker chromosome abnormality. Cellsuspensions of 5×10⁶ in 10 ml complete medium were incubated with 1.0 mlCa-precipitated DNA containing a total of 40 μg DNA as described byWigler et al., supra, for 4 hours at 37° C. in 5% CO₂ in tissue cultureflasks. For cells to be transformed to Mtx resistance, either 2 or 4 μgof DNA was from the 3T6R1 cell line. During this period differentiatedphagocytic marrow cells firmly adhered to the flask.

T6T6 cells were incubated with DNA from 3T6 R1 Mtx-resistant cells, andCBA/Ca marrow cells were incubated with control DNA preparations fromMtx-sensitive cells. Thereafter, loosely adherent cells were collectedand centrifuged at 150×g for 10 min and resuspended in DNA-free completemedium. After careful cell counts, Ca and T6T6 cells were combined in aratio of 1:1 and between 5×10⁶ and 5×10⁷ of the combined cells wereinjected intravenously into recipient CBA/Ca mice in a volume of 0.3 to0.4 ml in McCoy's medium with fetal calf serum. These recipients hadreceived 850 rads irradiation from the cobalt source 24 hours previouslyto eradicate endogenous hematopoiesis. This dose or irradiation wasselected because it had low lethality but virtually eradicatedendogenous spleen colonly-forming cells (CFU-S). Thus an average of 2±1endogenous CFU-S after 850 rads and 0.5±0.5 endogenous CFU-S after 900rad whole body irradiation was observed in this mouse strain. Between 48and 96 hours after injection, the recipient animals began treatment withthe previously established Mtx protocol.

Hematopoietic Effects of Methotrexate Treatment in the Mouse

An appropriate schedule of Mtx treatment which would select fordrug-resistant hematopoietic cells without lethality in control animalswas established as follows. Groups of normal CBA or C3H mice weighingbetween 18 and 25 g were treated by a thrice weekly schedule ofintraperitoneal injections of Mtx in doses varying between 0.5 and 8mg/kg per injection. An escalating schedule of 0.5 mg/kg for 4 doses, 2mg/kg for 4 doses and then 4 mg/kg thrice weekly was selected as notlethal but having profound suppressive effects on hematopoiesis. Tibialcellularity, peripheral white cell counts and hematocrits were alldepressed in Mtx-treated animals and megaloblastic morphologic changesdeveloped in the bone marrows. The hematocrit and tibial cellularitywere found to be the easiest and most reliable hematologic parameter tofollow and remained depressed in animals continuously treated with Mtxfor at least 3 months. False elevations of hematocrit in Mtx-treatedmice were occasionally observed in sick and dehydrated animals. Nodifference in sensitivity to Mtx was observed in the mouse strainsCBA/Ca and CBA/H T6T 6 as measured by standard hematologic parametersover 3 months of observation.

Selection of Drug Resistance Marrow Cells

The irradiated mice receiving mixtures of control Ca cells and T6T6cells transformed with 3T6 R1 DNA were treated with Mtx for periods of24 to 77 days. At intervals, animals were sacrificed or subjected to alimb amputation to obtain bone marrow samples. These were analyzed forkaryotype distribution, cellularity, CFU-S content and injected intosecondary irradiated CBA/Ca recipients. The results of two initialexperiments are shown in Tables I and II.

                  TABLE I                                                         ______________________________________                                        EXPERIMENT MB2. KARYOTYPE ANALYSIS OF                                         MARROW CELLS OF IRRADIATED CBA/Ca MICE                                        RECEIVING A 1:1 MIXTURE OF CONTROL Ca                                         and TRANSFORMED T6T6 MARROW CELLS                                                         Duration of Mtx Treatment                                                                       Karyotype                                       Recipient*  (days)            (% T6T6)                                        ______________________________________                                        Primary 1    0-24             57                                              Primary 2    0-32             59                                              Secondary 2 32-46             79                                              Primary 3    0-39             67                                              Secondary 3a                                                                              39-53             97                                              Secondary 3b                                                                              39-67             93                                              Secondary 3c                                                                              39-73             84                                              ______________________________________                                         *Irradiated CBA/Ca recipients of the 1:1 mixture of a Ca transformed with     wild type DNA and T6T6 cells transformed with 3T6R DNA are designated         "primary" and each mouse is given a unique number. The day of infusion is     designated "0." Recipients of marrow from "primary" animals are designate     "secondary" and bear the same identifying number. Karyotype analysis of       recipient bone marrow cells were performed after the designated interval      of methotrexate treatment. Between 50 and 100 chromosome spreads were         analyzed.                                                                

                  TABLE II                                                        ______________________________________                                        EXPERIMENT TV4. KARYOTYPE ANALYSIS OF                                         MARROW CELLS OF CBA/Ca MICE                                                   RECEIVING A 1:1 MIXTURE OF CONTROL Ca                                         and TRANSFORMED T6 MARROW CELLS                                                         Days with Mtx                                                                             Days Without                                            Recipient*                                                                              Mtx         (% T6)       Karyotype                                  ______________________________________                                        Primary 1 0-33        --           79                                         Primary 2 0-40        --           75                                         Primary 3 0-47        --           74                                         Primary 3 0-47        48-68        83                                         Secondary 3                                                                             47-61       --           88, 88, 100*                               Primary 4 0-54        --           75                                         Secondary 4                                                                             54-72       --           83                                         Primary 5 0-65        --           96                                         Primary 5 0-65         66-113      63                                         ______________________________________                                         *Irradiated recipients of the 1:1 mixture of Ca cells transformed with        wild type DNA and T6T6 cells transformed with 3T6R1 DNA are designated        "primary" and each mouse is given a unique number. The day of infusion is     designated "0." Recipients of marrow from "primary" animals are designate     "secondary" and bear the same identifying number.                             **Three secondary recipients. Between roughly days 30 and 40 a clear          increase in the percentage of bone marrow cells displaying the T6T6 marke     was observed in primary recipient animals. Marrow from these mice was         injected into irradiated secondary recipients which were then treated wit     methotrexate. They, too, showed an increased ratio of T6T6 to Ca              karyotypes, above that seen in the primary marrow recipients. Seven such      experiments were performed and this same pattern was seen in five             independent experiments involving 19 primary recipient animals and 30         secondary recipients. Only two experiments during this same period failed     to show a predominance of transformed karyotype.                         

When methotrexate treatment of animals receiving transformed marrowcells was stopped, the predominance of T6T6 karyotypes persisted for atleast 3 weeks (Primary Recipient 3, Table III) but gradually diminishedby 8 weeks without treatment (Primary Recipient 5, Table II).

                  TABLE III                                                       ______________________________________                                        KARYOTYPE ANALYSIS OF BONE MARROW AND                                         PLURIPOTENT STEM CELLS FROM CBA/Ca MICE                                       RECEIVING 1:1 MIXTURE OF CONTROL Ca AND                                       TRANSFORMED T6T6 BONE MARROW CELLS                                                              Bone Marrow Karyotype of                                                      Karyotype   Spleen Colonies                                         Duration of                                                                             T6T6        T6T6  Ca   Mixed                                Recipient                                                                             Mtx (days)                                                                              (%)         (%)   (%)  (%)                                  ______________________________________                                        Primary 1                                                                             0-24      57          50    50    0                                   Primary 2                                                                             0-40      75          57    26   17                                   Primary 3                                                                             0-47      74          58     8   33                                   ______________________________________                                         Individual spleen colonies were removed 10 days after innoculation of         irradiated recipient with bone marrow cells. A single cell suspension was     made from each colony and cells were incubated with colcemide 3 μglml      for 90 minutes before treatment with hypotonic KCL and fixation with          acetic acid/ethanol for chromosome spreads.                              

In order to analyze whether the predominance of T6T6-marked cellsinvolved pluripotent stem cells as well as other proliferating marrowcells, marrow was taken from selected primary recipient animals and5×10⁴ cells were injected into irradiated recipient CBA/Ca mice in atypical spleen colony-forming (CFU-S) assay. (Tell and McCulloch Rad.Res. 14:213, 1961) Ten days later the secondary recipients were killedand individual spleen colonies removed for karyotype analysis. As seenin Table III the percentage of T6T6 karyotype predominated in thepluripotent marrow stem cell population. Mixed T6T6-Ca spleen colonieswere also seen, presumably resulting from development of T6T6 colonieson a background of endogenous hematopoiesis in the Ca animals.

Effect of Drug Administration on Cell Predominance

In order to assess the significance of these results, controlexperiments were performed to determine whether T6T6-marked cells hadany proliferative advantage or increased resistance to Mtx and toanalyze the contribution of endogenous hematopoietic repopulation inirradiated CBA/Ca animals. Experimental animals receiving an equalmixture of mock transformed Ca and mock transformed T6T6 and eitheruntreated or treated with Mtx for up to two months had a predominance ofCa karyotypes as anticipated from the contributions of infused Ca cellsand endogenous Ca cells.

                  TABLE IV                                                        ______________________________________                                        KARYOTYPE ANALYSIS OF MARROW CELLS OF                                         CONTROL Ca MICE RECEIVING A 1:1 MIXTURE                                       OF MOCK TRANSFORMED Ca AND MOCK                                               TRANSFORMED T6T6 MARROW CELLS                                                             Duration of Mtx Treatment                                                                       Karyotype                                       Recipient*  (days)            (% T6T6)                                        ______________________________________                                        Primary 1   0-33              31                                              Primary 2   0-40              24                                              Primary 3   none              26                                              Primary 4   0-60              40                                              Primary 5   none              21                                              Primary 6   0-26              50                                              Secondary 6 27-48             28                                              Primary 7   0-53              56                                              Primary 8   0-56              57                                              Primary 9   0-42              40                                              Primary 10  0-56              24                                              ______________________________________                                    

In primary Ca recipients of equal mixtures of mock transformed T6 and Cacells the percentage of dividing marrow cells with the T6 marker variedbetween 21 and 57%. It is presumed that animals with lower percentagesof T6 had restored their hematopoiesis at least in part from endogenousCa cells surviving the irradiation.

In a final study to demonstrate transformation to drug resistance, intwo independent experiments the usual procedure was reversed and Cacells were transformed and T6 cells were used as the controls. Afterinjection of a 1:1 mixture of Ca and T6 into irradiated T6 animals theywere treated with Mtx or left untreated for two months.

                  TABLE V                                                         ______________________________________                                        EXPERIMENT MB6. KARYOTYPE ANALYSIS OF                                         MARROW CELLS CBA/T6T6 MICE RECEIVING A                                        1:1 MIXTURE OF CONTROL T6 CELLS AND                                           TRANSFORMED Ca MARROW CELLS                                                                   Mtx         Duration                                                                             Karyotype                                  Recipient                                                                              Exp    Treatment   (days) (% Ca)                                     ______________________________________                                        Primary 1                                                                              1      Yes         42     55                                         Primary 2                                                                              1      None        59     33                                         Primary 3                                                                              1      Yes         59     62                                         Primary 4                                                                              1      Yes         71     68                                         Primary 5                                                                              1      None        71     35                                         Primary 6                                                                              1      Yes         97     72                                         Primary 1                                                                              2      Yes         56     67                                         Primary 2                                                                              2      None        56     40                                         ______________________________________                                         Recipient T6 mice received aliquots of a 1:1 mixture of T6 cells              transformed with wild type DNA and Ca cells transformed with 3T6 R1 DNA.      Animals were either untreated or treated with Mtx.                       

As shown in Table V, the Ca karyotype predominated only when Mtx wasadministered. This gave an unambiguous demonstration that drug therapydetermined the predominant marrow population and that the karyotype perse did not influence predominance; i.e., there was no inherently greaterresistance to Mtx associated with either the CBA/Ca strain or theCBA/HT6T6.

Hematologic Status of Mice Receiving Transformed Marrow

After it was demonstrated that cells transformed with Mtx-resistant DNApredominated in the marrows of drug-treated recipient animals, simpletests were performed to assess the animals' hematologic status.

                  TABLE VI                                                        ______________________________________                                        HEMATOLOGIC STATUS OF MICE                                                    RECEIVING TRANSFORMED BONE MARROW*                                                      Mtx                                                                           treat-  CFU-S                                                                 ment    (per 5 ×                                                                          Tibial   Hematocrit                               Recipients                                                                              (days)  10.sup.4 cells)                                                                         Cellularity                                                                            (%)                                      ______________________________________                                        Controls: saline-                                                                       none    11.3 (5-15)                                                                             8.7 (7.1-10.6)                                                                         42 (38-47)                               treated (15)                                                                  Controls: Mtx-                                                                          21-77   --        3.9 (3.3-4.9)                                                                          26 (23-39)                               treated (15)                                                                  Primary 1 0-33    8.8 (7-9) 13.9     --                                       Primary 2 0-40    16 (13-19)                                                                              9.6      --                                       Primary 3 0-47    22 (18-25)                                                                              6.0      --                                       Primary 4 0-54    --        9.6      --                                       Secondary 4                                                                             54-72   --        6.6      40                                       Primary 5 0-65    --        8.2      47                                       ______________________________________                                         *Control animals received no irradiation or bone marrow cells and were        injected with saline or Mtx as described. Number of animals is indicated      in parentheses in column one. Numbers in parentheses in other columns         indicate the range of values observed. Primary recipient CBA/Ca mice were     irradiated (850 rads) and received a 1:1 mix of control Ca and transforme     T6 cells before treatment with Mtx for the periods indicated. Secondary       recipients were irradiated and received marrow from primary recipients.       CFUS were assayed by injecting 5 × 10.sup.4 marrow cells into           irradiated CBA/Ca recipient mice and counting spleen colonies 10 days         later. (Tall and McInttosh, supra)                                       

The results displayed in Table VI indicate that animals receivingtransformed bone marrow and methotrexate have high hematocrits andtibial cellularity relative to control animals receiving methotrexatebut no transforming DNA. Further, they have high levels of CFU-S similarto control animals which received neither radiation nor bone marrowcells. Similar results were seen in other independent experiments. Thus,the hematologic status of mice receiving transformed bone marrow treatedwith 3T6 R1 DNA in vitro returned toward normal despite persistenttreatment with methotrexate. Autopsies were performed on these animalsat intervals up to 150 days after transformation and sections ofvisceral organs were taken for microscopic review. These studiesrevealed no abnormalities and the animals were clinically well at thetime of sacrifice.

DHFR (Dihydrofolate Reductase) Assay

To assess whether drug resistance was related to higher enzyme levels inmice receiving transformed bone marrow, spleens were removed fromprimary, secondary or tertiary recipients of transformed marrow fromfour independent experiments and assayed for DHFR. These recipient micewere initially treated with Mtx and treatment was terminated 5-7 daysprior to the collection of tissue for the DHFR assays. Appropriatecontrols from 12 syngeneic animals either irradiated or not, anduntreated or treated with Mtx for periods up to six weeks, were alsoused as spleen donors. In each case, conditions for control animals werechosen to match those of the experimental group. A radiometric assay forDHFR was performed on sonicated cell-free extracts of spleen. (SeeHillcoat et al., Proc. Natl. Acad. Sci. (USA) 58:1632, 1967; Hayman etal. Anal. Biochem. 87:460, 1978).

                  TABLE VII                                                       ______________________________________                                        DIHYDROFOLATE REDUCTASE ACTIVITY IN                                           SPLEEN AND KARYOTYPE ANALYSIS OF BONE                                         MARROW IN CBA/Ca RECIPIENTS RECEIVING                                         1:1 MIXTURE OF CONTROL Ca AND                                                 TRANSFORMED T6T6 BONE MARROW CELLS*                                                               Bone Marrow                                                       Duration of Karyotype                                                         Mtx         Predominance                                                                              Spec. activity                                Recipient                                                                             (days)      (%)         (% Control)                                   ______________________________________                                        Primary  0-42       49          168                                           Primary  0-59        62**       375                                           Secondary                                                                             39-67       93          391                                           Secondary                                                                             54-72       83          183                                           Secondary                                                                              65-107     --          208                                           Tertiary                                                                              58-80       --          224                                           ______________________________________                                         *Spleen fragments were sonicated for 30 seconds in the cold and assayed       for DHFR by the method of Hayman et al Anal. Biochem. 37:460,1978. Result     are expressed as the precent of DHFR in spleens of simultaneously             sacrificed control mics.                                                      **This animal received transformed Ca cells and had Ca karyotype              predominance. All others received transformed T6T6 cells.                

DHFR-specific activity was approximately 2- to 4-fold greater in thespleen extracts of animals receiving transformed bone marrow than incontrols. These were considered to be underestimates of the elevationsof DHFR in the hematopoietic cells of animals receiving transformedmarrow, since the extracts of spleen also included stromal and capsulartissues and non-proliferating lymphoid cells.

Transformation of tk⁺ and tk⁻ L Cells in Vitro

The technique of Wigler et al., supra, was employed to insert herpesvirus tk gene in tissue culture cells. A Bam H1 fragment containingHSVtk gene was purified by gel electrophoresis and phenol extractionfrom Sea-Plaque® agarose gel. Concatemers of the fragment wereconstructed using T4 ligase. Mouse L cells lacking thymidine kinase(tk⁻) were incubated with a calcium phosphate precipitate of highconcentrations of DNA containing the herpes tk gene in the plasmidvector pBR 322. After a brief period of incubation, cells weretransferred to a selected medium favoring the growth of tk⁺ cells. Tk⁻cells transformed to tk⁺ status were selected in HAT medium which favorsthe growth of tk⁺ cells. The principle underlying these experiments isthat tk⁺ cells can rescue themselves from the block in thymidinesynthesis imposed by aminopterin in HAT medium by utilizing exogenousthymidine, whereas tk⁻ cells lacking the kinase cannot. Tk⁺ clones willgrow under these selective conditions. The anticipated results wereobserved.

                  TABLE VIII                                                      ______________________________________                                        THYMIDINE KINASE SPECIFIC ACTIVITY OF                                         MOUSE CELLS TRANSFORMED IN VITRO                                              WITH HERPES SIMPLEX VIRUS TK DNA*                                                                               Specific                                                         Transformation                                                                             Activity                                    Clone    Cell Lineage                                                                              with         (cpm/μg)                                 ______________________________________                                        --       Ltk.sup.-    --          0.08                                        --       Ltk.sup.+  (wild                                                                           --          4.9-5.5                                              type)                                                                207      Ltk.sup.-   HSVtk in     9.1                                                              plasma                                                   202                                                                           205      Ltk.sup.+   HSVtk in     10.3                                                             plasmid (3)                                              214      Ltk.sup.+   HSVtk in     12.3                                                             plasmid (3)                                                                   Sal cut and                                                                   ligated (3)                                              214-3    Ltk.sup.+                22.                                         ______________________________________                                         *Clones were derived from the parent cell line by transformation with         calcium precipitated HSV plasmid DNA. Clones of Ltk.sup.-  lineage were       selected for and grown in HAT medium.                                         Cells of NCTC (wild type) lineage were selected for in medium containing      10.sup.-4 M MTX, 0.1 M thymidine and grown up in HAT medium.             

Surprisingly, thymidine kinase activity of different clones varied overa considerable range, suggesting multiple expressed copies of tk gene insome transformants.

In addition to transforming tk⁻ cells to tk⁺ status, wild type tk⁺ cellswere transformed in such a manner that they contained additional copiesof the tk gene of viral origin. Wild type tk⁺ cells will grow in HATmedium by utilizing the available thymidine and HAT medium could not beused to distinguish wild type cells from those incorporating additionalcopies of viral genes. Consequently, a range of methotrexate andthymidine concentrations were explored which would inhibit normal cellswith a single copy of tk gene but allow cells with increasedconcentrations of tk to grow. These selective conditions required higherfolate antagonist concentrations (methotrexate 10⁻⁴ M) and lowerthymidine levels (0.1 μg/ml) than used in conventional HAT medium.

Wild type (tk⁺) L cells were exposed to herpes virus tk DNA undertransforming conditions and then cultured under selective conditions. Anumber of transformed clones were isolated, and some were grown tosufficient density to allow for measurement of tk-specific activity(Table VIII) and for analysis of tk-specific gene sequences in DNA bySouthern hybridization. For convenience, those wild type tk⁺ cellstransformed with herpes tk gene and containing at least one viral genein addition to the mammalian tk gene will be designated as tk⁺⁺.

Selection of tk Transformed Cells In Mice

The strategy for selection of cells transformed to tk⁺⁺ was similar tothat employed for selection of expression of the DHFR gene and isillustrated in FIG. 2. ##STR1## Experimental design for selecting invivo cells transformed to Mtx resistance by calcium-precipitated HSVtkDNA. Donor T6T6 and Ca mice were pretreated with 3 mg vinblastine 3 daysprior to in vitro transformation with DNA from HSVtk and salmon spermrespectively. Transformed T6 and Ca cells in a mixture of 1 to 1.5 wereinjected into irradiated Ca recipients, some of which were subsequentlytreated with Mtx, karyotype analysis of marrows from recipients was doneto show predominance of T6T6 cells induced to Mtx resistance.

Mouse bone marrow cells with a distinctive chromosomal marker (T6T6)were obtained from intact normal animals and treated in vitro with acalcium microprecipitate of herpes virus tk gene. The treated marrow,presumed to contain a few stem cells transformed by viral gene, wasmixed in a ratio of 1:1.5 with "mock" transformed syngeneic marrow cellslacking the chromosomal marker (CBA/Ca cells). These Ca cells had beenincubated under transforming conditions with control DNA from salmonsperm. The cell mixture was injected into genetically compatible CBA/Caanimals that had been irradiated in order to greatly reduce endogenoushematopoiesis. The infused cells gradually restored blood cell formationin marrow-depleted recipients. During the period of reconstitution someof the animals were treated with methotrexate to allow for the selectiveproliferative advantage for those transformed cells that hadincorporated one or more copies of a functional viral tk gene.Transformed cells were identified by their distinctive chromosomalmarker. If there were no selective advantage to transformed cells, thenthe ratio of marked to unmarked dividing hematopoietic cells would beless than 1:1.5; if, however, there were a proliferative advantage, thenmarked cells with increased levels of tk would be found to constitutemore than 50% of the dividing marrow population.

In addition to this indirect means of detecting a population ofhematopoietic cells transformed to methotrexate resistance, DNAextracted from the spleens of transformed and control mice was alsoanalyzed for the presence of sequences unique to the viral tk gene.

Simultaneous control studies were performed in which some recipients ofthe mixture of transformed T6 and mock transformed Ca cells were leftuntreated by methotrexate in order to assess the contribution ofselective pressure by drug. In other control studies, equal numbers ofuntransformed T6 and Ca cells were injected into Ca recipients, some ofwhom were treated with methotrexate and others were left untreated. Infurther control studies, Ca marrow cells were transformed with herpesvirus tk and mixed with mock transformed T6 cells. These mixtures werethen injected into irradiated CBA/T6T6 animals which were subsequentlyexamined for the predominance of Ca karyotypes in proliferating bonemarrow.

The T6 marker was selected because of its ready identification and theavailability of syngeneic animals possessing and lacking this marker.CBA/H-T6T6 mice have the chromosomal anomaly, whereas compatible CBA/Camice lack the marker. Bone marrow was selected for transformationbecause of its accessibility, high rate of proliferation, and thepersistence of pluripotent stem cells throughout adult life. Of all thecell types present in the marrow, only transformation of appropriatestem cells should lead to the establishment of long-term hematopoieticactivity in which cells bearing viral DNA sequences persisit.

The technique of transformation was as follows. Equal numbers of CBA/Caand CBA/T6T6 mice were injected intraperitoneally with 3 mg/kg of themitotic inhibitor, vinblastine, three days before isolating cells fortransformation. Mitotic inhibition by this treatment is followed by aburst of stem cell proliferation. Marrow cells from vinblastine-treatedanimals were relatively depleted of mature cells and enrichedapproximately threefold in pluripotent spleen-colony-forming cells(CFU-S) (Smith et al., J. Nath. Cancer Inst. 40: 847-854, 1968; Till andMcCulloch, Rad. Res. 14: 213, 1961) On the day of transformation,single-cell suspensions in McCoy's 5A medium with 15% fetal calf serumwere obtained from femurs and tibias of sacrificed animals. Cells fromCa and T6 animals were placed in separate pools. Cell suspensions at5×10⁷ in 10 ml of complete medium were incubated with 1 mlcalcium-precipitated DNA containing 8 μg herpes virus tk in plasmid formas well as purified HSVtk Bam H1 fragment (without plasmid) ligated toform concatemers. Incubation was continued for 4 hours at 37° C. in 5%CO₂. During this period some marrow cells adhered to the flask. Asimilar procedure was used for Ca cells which were incubated with salmonsperm DNA. Loosely adherent cells were collected after incubation andcentrifuged at 150×g for 10 minutes, then resuspended in DNA-freecomplete medium. After careful cell counts, T6 and Ca cells werecombined in a ratio of 1:1.5 and between 2×10⁶ and 1×10⁷ cells wereinjected intravenously into recipient CBA/Ca mice that had beenirradiated 24 hours previously with 850 rads. This dose of irradiationvirtually eliminated endogenous spleen colony formation. Between 72 and96 hours after injection, the recipient animals began treatment with apreviously established methotrexate protocol. An escalating schedule of0.5 mg/kg thrice weekly for four doses, 2 mg/kg for four doses, and then4 mg/kg thrice weekly was selected as nonlethal but having profoundsuppressive effects on hematopoiesis. Tibial cellularity, peripheralwhite counts, and hematocrits were all depressed in methotrexate-treatedanimals. The mice receiving mixtures of control Ca cells and transformedT6 cells were treated with methotrexate for a period of 28 to 70 days.At intervals, animals were sacrificed or subjected to a limb amputationto obtain marrow samples, which were analyzed for karyotype distributionand cellularity and injected into secondary recipient animals.

The results of one such experiment are shown in Table IX. By day 32after transformation, a clear predominance of T6-marked cells in adividing marrow population of a methotrexate-treated animal wasobserved. Control animals receiving an equal mixture of untransformed T6and Ca cells (1:1) had a predominance of Ca karyotype. In these controlrecipients a ratio of Ca to T6 karyotype of greater than 1 wasanticipated because of the contribution of endogenous CBA/Cahematopoiesis in these recipient animals. In 20 such control animalsnone showed a predominance of the T6 karyotype.

                  TABLE IX                                                        ______________________________________                                        KARYOTYPE ANALYSIS OF MARROW CELLS FROM                                       CBA/Ca MICE RECEIVING A 1:1.5 MIXTURE OF                                      tk TRANSFORMED T6T6 AND CONTROL Ca CELLS                                                Duration                                                                      of Mtx                 Detectable                                             Treatment Karyotype    viral tk                                     Recipient (days)    (% T6 Cells) DNA sequences                                ______________________________________                                        Primary 1  0-32     74           Positive                                     Primary 2  0-47     84           N.T.                                         Primary 3 No Mtx**  35           N.T.                                         Primary 4 47-60     N.T.         Positive                                     Secondary 4                                                                             60-89     17           Negative                                     Controls (20)                                                                            0-90     38 ± 10   Negative                                     ______________________________________                                         *Irradiated CBA/Ca mice received aliquots of a 1:1.5 mixture of T6 cells      transformed with herpes virus tk plasmid and ligated herpes virus tk gene     and Ca cells mocktransformed with salmon sperm DNA. These mice are            designated "primary." Recipients of marrow from primary animals are           designated secondary and bear the same identifying number.                    **Primary animal #3 was not treated with Mtx and was followed for 47 days     N.T. = not tested.                                                       

As seen in Table IX, only the methotrexate-treated primary recipientanimals showed a predominance of the T6 karyotype. An untreated animaland secondary recipients of marrow from primary recipients demonstratedpredominance of the Ca karyotype. Similar results were seen in threeother experiments in which animals were followed for 60 days. In one ofthese experiments Ca cells were transformed and T6 cells served ascontrols.

Herpes Simplex Virus Sequences In Mouse Hematopoietic Cells

To unambiguously demonstrate insertion of viral genes into proliferatingmarrow cells, DNA from spleens of experimental and control mice wasextracted and subjected to Southern blot analysis. (Southern, J. Mol.Biol. 98: 503, 1975) DNA from these sources was cut with the restrictionenzyme Eco/R1 and transferred to nitrocellulose strips after agarose gelelectrophoresis. The strips were incubated with P³² -labeled herpesvirus Bam H1 fragment containing DNA sequence coding for tk underhybridizing conditions and then analyzed by autoradiography. As shown inTable 2, herpes virus sequences were demonstrable in spleen DNA ofprimary recipient mice receiving bone marrow transformed in vitro andselected by drug pressure in vivo. Control mice and secondary recipientmice from the same experiment but lacking T6 predominance failed todemonstrate herpes tk sequences.

The transfer of genes for drug resistance to hematopoietic cells invitro and their selection in intact animals in vivo provides for avariety of clinical applications. Such applications include the transferof drug resistance genes with the objective of enabling patients withcancer to tolerate higher doses of anti-neoplastic drugs and insertionof genes which confer a proliferative advantage coupled to other genesto treat human genetic diseases such as the hemoglobinopathes.

In the past, supression of bone marrow hematopoiesis has been a majorlimitation to intensive treatment with most anticancer chemotherapeuticdrugs. By enhancing resistance of marrow cells, higher dosages may bepermitted in the treatment of cancers of other tissues. The above datahave demonstrated that mice receiving marrow transformed withMtx-resistant DNA tolerate high doses of Mtx for long periods of timewith nearly normal hematologic parameters. With the hemoglobinopathies,insertion of a normally regulated and structurally normal β-globin geneshould be capable of correcting the defect in β-thalassemia and sicklecell disease. By using the normally regulated and structurally normalβ-globin gene in conjunction with a selective marker to allow forselective pressure in the host, the cells containing the selectivemarker and structurally normal β-globin gene should proliferate underthe selective pressure of drug treatment.

The subject invention can be employed with host cells having adeficiency which results in a low rate of proliferation. By introductionof DNA providing for correction of the deficiency, the modified cellswould have a proliferative advantage over unmodified cells of the sametype. This situation may be illustrated by cases of combinedimmunodeficiency disease in which there is an adenosine deaminasedeficiency.

The subject invention demonstrates that with appropriate selectiveconditions, naturally occurring or stress induced, modified cells underproliferating conditions can be given a selective advantage over theunmodified host cell. Thus, mammalian hosts can be provided withdifferent or enhanced capabilities as a result of or concurrent withintroduction of DNA providing for a selective advantage.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A method for enhancing the genetic capability ofa mammalian host by employing mammalian cells having exogenous geneticmaterial capable of expression which comprises:isolating regenerativemammalian parent cells from a mammlian source compatible with said host;combining said regenerative mammalian parent cells with DNA including atleast one gene capable of providing a selective advantage over theparent cell due to a naturally occurring deficiency or external stressinducement, said combining being under conditions where said DNA isintroduced into said parent cell to produce modified cells; andintroducing said modified cells into a compatible mammalian host undernaturally occurring deficiency conditions or external stress inducedconditions in said host providing said selective advantage, whereby saidmodified cells regenerate in said host at the source of said cells.
 2. Amethod according to claim 1, wherein said parent cells are in thedividing phase.
 3. A method according to claim 1, wherein said one geneproviding a selective advantage expresses an enzyme.
 4. A methodaccording to any of claims 1, 2 or 3, wherein said parent cells are bonemarrow stem cells.
 5. A method according to claim 3, wherein said enzymeis inhibited by a chemotherapeutic drug and said external stress inducedconditions in said host comprise administering to said host saidchemotherapeutic drug.
 6. A method according to claim 5, wherein saiddrug is methotrexate.
 7. A method according to claim 1, including theadditional step of isolating said modified cells from said host.